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Title Neuropeptide Y activates phosphorylation of ERK and STAT3 in stromal vascular cells from brown adipose tissue, butfails to affect thermogenic function of brown adipocytes
Author(s) Shimada, Kohei; Ohno, Yuta; Okamatsu-Ogura, Yuko; Suzuki, Masahiro; Kamikawa, Akihiro; Terao, Akira; Kimura,Kazuhiro
Citation Peptides, 34(2), 336-342https://doi.org/10.1016/j.peptides.2012.02.012
Issue Date 2012-04
Doc URL http://hdl.handle.net/2115/49225
Type article (author version)
File Information Pep34-2_336-342.pdf
Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP
1
Neuropeptide Y activates phosphorylation of ERK and STAT3 in stromal vascular
cells from brown adipose tissue, but fails to affect thermogenic function of brown
adipocytes
Kohei Shimada1, Yuta Ohno
1, Yuko Okamatsu-Ogura
1, Masahiro Suzuki
1, Akihiro
Kamikawa1,2
, Akira Terao1, and Kazuhiro Kimura
1
1Department of Biomedical Sciences, Graduate School of Veterinary Medicine,
Hokkaido University, Sapporo, Japan
2Department of Basic Veterinary Medicine, Obihiro University of Agriculture and
Veterinary Medicine, Obihiro, Hokkaido, Japan
Running title: NPY in BAT
Corresponding author: Kazuhiro Kimura, Ph.D.
Department of Biomedical Sciences, Graduate School of Veterinary Medicine,
Hokkaido University, Sapporo 060-0818, Japan
Tel. & Fax: +81-11-757-0703, E-mail: [email protected]
2
Abstract
The thermogenic function of brown adipose tissue (BAT) is increased by
norepinephrine (NE) released from sympathetic nerve endings, but the roles of NPY
released along with NE are poorly elucidated. Here, we examined effect of NPY on
basal and NE-enhanced thermogenesis in isolated brown adipocytes that express Y1 and
Y5 receptor mRNA. Treatment of cells with NPY did not influence the basal and
NE-enhanced rates of oxygen consumption and cAMP accumulation. Treatment with
NPY also failed to induce ERK (Thr202/Tyr204) phosphorylation in the brown
adipocytes. In contrast, treatment with NPY increased ERK phosphorylation in cultured
stromal vascular cells from the BAT that express Y1 receptor mRNA. In the latter
treatment with NPY also increased STAT3 (Ser727) phosphorylation. These results
suggest that NPY mainly acts on stromal vascular cells in BAT and plays roles in the
regulation of their gene transcription through ERK and STAT3 pathways, while NPY
does not affect the thermogenic function of brown adipocytes.
Key words: BAT, norepinephrine, p44/p42 MAPK, NPY, STAT, thermogenesis
3
1. Introduction
Acute cold exposure and feeding significantly stimulate thermogenesis in the
brown adipose tissue (BAT) [6, 9, 25, 33, 35]. The sympathetic nervous system (SNS)
controls this adaptive thermogenesis through the activation of -adrenergic receptors
(ARs), especially 3AR. It is well established that ARs are coupled to the
stimulatory G-protein (Gs) and thereby sequentially activate adenylyl cyclase (cAMP
formation) and cAMP-dependent protein kinase (PKA). It is also established that active
PKA leads to activation of lipolytic enzymes and enhanced expression of the genes
encoding uncoupling protein-1 (UCP1). Furthermore, thermogenesis in the BAT is
principally dependent on the activation of UCP1, which facilitates proton leakage
across the inner mitochondrial membrane to dissipate the electrochemical gradient as
heat.
The co-existence of sympathetic and peptidergic innervation has been
demonstrated in BAT [5, 11, 23, 28, 38]. Among peptidergic neurotransmitters,
neuropeptide Y (NPY) levels in the BAT are significantly decreased and increased by
surgical excision of the sympathetic nerves to the BAT and putting an animal into a cold
environment, respectively [30]. One study suggested the presence of two separate
subpopulations of autonomic nerves in the BAT, that is, one fiber containing both NE
and NPY innervates the vascular system, and the other fiber containing only NE
innervates the brown adipocytes [5, 6]. Similarly, it is suggested that NPY co-localizes
with tyrosine hydroxylase in noradrenergic neurons, but only in the perivascular
nerve fiber network [11]. However, other studies showed that NE- and NPY-positive
fibers were also found around parenchymal brown adipocytes in mice [38] and rats [23,
28].
4
NPY exhibits its physiological effects through at least four receptors known as Y1,
Y2, Y4, and Y5. It is well established that NPY receptors are coupled to the inhibitory
G-protein (Gi) and thereby antagonize -adrenergic agonist-induced activation of
adenylyl cyclase [27, 36]. Moreover, NPY potentiates NE actions via -adrenoceptor
[18, 36, 37], and the potentiation is attributed to the suppression of -adrenergic action
[18]. In the BAT, NPY is suggested to have a vasoconstrictor role at arteriovenous
anastomoses [40] and a suppressive role in BAT thermogenesis [30]. However, when
treated with NPY and NE in combination for 14 days, rats increase their minimal
oxygen consumption by approximately 38% compared with vehicle controls [2]. Similar
phenomena are observed in cold acclimated rats [3]. However, direct effects of NPY on
BAT thermogenesis have not been elucidated yet.
Therefore, in the present study, we first examined the effects of NPY on brown
adipocytes expressing Y1 and Y5 NPY receptors. We also examined the effects of NPY
on stromal vascular cells (SVC) expressing Y1 NPY receptor.
5
2. Materials and methods
2. 1. Materials
Dulbecco’s modified Eagle’s medium (DMEM) and collagenase were purchased from
Wako Pure Chemicals Co. (Osaka, Japan). Fatty acid-free bovine serum albumin (BSA)
and isobutylmethylxanthine (IBMX) were purchased from Sigma-Aldrich Fine
Chemicals (St. Louis, MO, USA). Fetal calf serum (FCS) was obtained from Trace
Scientific Ltd. (Melbourne, Australia). The following antibodies were purchased from
Cell Signaling Technology (Beverly, MA, USA): anti-phospho-specific ERK1/2
(p44/42MAPK, Thr-202/Tyr-204) antibody, anti-ERK1/2 antibody,
anti-phospho-specific STAT3 (Ser-727) antibody, anti-phospho-specific STAT3
(Tyr-705) antibody, and anti-STAT3 antibody.
2.2. Animals
Experimental procedures and animal care were carried out in accordance with the
Guidelines of Animal Care and Use from Hokkaido University, and were approved by
the University Committee for the Care and Use of Laboratory Animals. Male
8-week-old C57BL/6J mice were obtained from Nihon SLC (Shizuoka, Japan), housed
under specific pathogen-free conditions at 24°C with a 12 h:12 h light:dark cycle, and
given food and water ad libitum.
2.3. Isolation of brown adipocytes and stromal vascular cells (SVC)
Mice were anesthetized with an intraperitoneal injection of pentobarbital (50 mg/Kg).
The whole body was perfused first with Ca2+
- and Mg2+
-free PBS and then with
Krebs-Ringer bicarbonate HEPES buffer (KRBH, pH 7.4) containing collagenase (1
6
mg/ml), through a cannula inserted into the left ventricle with a drain from the right
atrium in an open-chest procedure. Interscapular BAT was then rapidly removed and
placed in PBS. After careful removal of extraneous tissue, the BAT of two mice was
transferred in KRBH containing fatty acid–free BSA (10 mg/ml), 2.5 mM glucose, and
collagenase (1 mg/ml), minced well, and incubated for 30 minutes at 37 °C with
shaking at 90 cycles/min. The cell suspension was passed through a 200-m nylon filter
and the filtrate was centrifuged at 200 g for 1 min at room temperature. The floating
cells were collected as brown adipocytes, and washed 3 times with KRBH to eliminate
collagenase. The cells were diluted to a density of 3-5 × 105 cells/ml for BAT with the
assay buffer [KRBH containing fatty acid–free BSA (40 mg/ml) and 2.7 mM glucose].
The cell suspensions were kept for 1 h at room temperature before analysis. The
remainder after the collection of adipocytes was centrifuged at 1,200 g for 10 min at
room temperature. The supernatant was removed and the pellet was suspended and
cultured in DMEM containing 10% FCS in collagen-coated dishes (Iwaki-Asahi Techno
Glass, Chiba, Japan), and the media were changed every 2 days. Cells between the first
and second passages were used for the experiments as stromal vascular cells (SVC).
2.4. Measurement of oxygen consumption in isolated brown adipocytes
Oxygen consumption of isolated adipocytes was measured polarographically with a
Clark-style oxygen electrode in a water-jacketed Perspex chamber at 37 °C [29]. The
cell suspension (200 μl) was added to a magnetically stirred chamber set with a
thermostat at 37°C and was adjusted to a final volume of 1 ml with the assay buffer.
The chamber was closed, and the cells were incubated for 5 min to determine the basal
respiratory rate. NE and/or NPY were then injected with a Hamilton syringe through a
7
small hole in the cover of the chamber. Oxygen concentration in the chamber was
monitored for 15 min. Oxygen consumption rates were calculated using a computer
program (782 System, Strathkelvin Instruments, Glasgow, Scotland, UK).
2.5. cAMP accumulation in isolated brown adipocytes
Isolated adipocytes (3 x 105
cells) were incubated in 0.9 ml of the assay buffer
containing 0.5 mM IBMX (a phosphodiesterase inhibitor), and stimulated with NE (1
M) and/or NPY (0.1 M) for 10 min. HCl (0.1 ml, 1N) was then added and samples
were kept on ice for 10 min before centrifugation at 15,000×g for 15 min at 4 °C. The
cAMP content in the supernatant was then measured using a cyclic AMP Assay kit
(Enzo Life Sciences International, Inc., Plymouth Meeting, PA, USA), according to the
protocol provided.
2.6. ERK activation in isolated brown adipocytes
Isolated adipocytes (5 x 104 cells) were incubated in 0.1 ml KRBH, and stimulated with
NE (1 M) and/or NPY (0.1 M) for 10 min. HCl (10 µl, 1N) was then added and
samples were kept on ice for 10 min before adding chloroform/methanol (1:2, 0.5 ml).
The solution was centrifuged at 15,000×g for 1 min at 4 °C, and the resultant pellet was
washed once with chloroform/methanol and solubilized in a sample buffer for
SDS-PAGE.
2.7. Western blotting
Aliquots of cellular protein as described above (20 g) were separated by SDS-PAGE
and the proteins were transferred onto PVDF membranes (Immobilon; Millipore,
8
Bedford, MA, USA). The membranes were incubated in a blocking buffer [20 mM
Tris/HCl (pH7.5)/150 mM NaCl] containing 0.1% Tween 20 and 1% skimmed milk,
and then in the buffer containing a primary antibody. The bound primary antibody was
visualized using horseradish peroxidase-linked goat anti-rabbit IgG (Zymed
Laboratories, South San Francisco, CA, USA) and chemiluminescent HRP Substrate
(Millipore) according to the manufacturer’s instructions. The intensity of
chemiluminescence for the corresponding bands was analyzed using Image J, a
public-domain image-processing and analysis program.
2.8. Cell culture and treatments
SVC were cultured onto 100 mm dishes in DMEM supplemented with 10% fetal bovine
serum in 5% CO2 in humidified air at 37°C. After cells were grown to confluence, they
were cultured in a serum-free medium for 24 h to render them quiescent. The cells were
then treated with NPY and vehicle (PBS) for 10 min at 37°C. Subsequently, the cells
were washed once with buffer [50 mM Hepes (pH7.5), 150 mM NaCl, 5 mM EDTA, 10
mM sodium pyrophosphate, 2 mM NaVO3, protease inhibitor mixture (Complete;
Roche Diagnostics, GmbH, Germany)], then lysed with the buffer containing 1%
Nonidet P-40. The lysate was kept on ice for 30 min and centrifuged at 15,000g for 15
min at 4°C. The resulting supernatant was stored at -30°C. Proteins were determined by
the Lowry method using BSA as a standard [26]. When treated, PD98059 (an ERK
kinase inhibitor; EMD Biosciences, Inc., La Jolla, CA, USA) was added to the cultured
cells 2 h before NPY stimulation. Aliquots of the cell lysate were analyzed by Western
blotting.
9
2.9. Cell proliferation assay
SVC (5 × 103 cells) were cultured in 96-well plates with DMEM containing 1% FCS
and increasing concentrations of NPY for 72 h. Then, the number of cells was assessed
using Cell Counting Kit-8 (DOJINDO, Kumamoto, Japan).
2.10. mRNA analysis
Total cellular RNA was isolated from isolated adipocytes, cultured cells and mouse
tissues by the guanidine-isothiocyanate method using RNAiso reagent (Takara Bio,
Shiga, Japan). First, 2 µg of total RNA was mixed with DNaseI reaction buffer [DNaseI
(Roche), 40 mM Tris-HCl (pH7.2), 6 mM MgCl2, ribonuclease inhibitor (Takara)]. The
reaction was carried out at 37 °C for 30 min, and terminated at 95 °C for 5 min. Then,
single-strand cDNA was synthesized using 100 units of Moloney murine leukemia virus
reverse transcriptase (Invitrogen Co., Carlsbad, CA, USA), 50 pmol poly(dT) primer,
and 20 nmol dNTP in a total volume of 20 µl at 37 °C for 1 h. The reaction was
terminated at 95 °C for 5 min.
The expression of mRNA was analyzed by conventional PCR or real-time PCR using
the cDNA as a template. PCR amplification was performed with 2.5 units Taq
polymerase (Ampliqon, Herlev, Denmark), 3 mM MgCl2, and 50 pmol forward and
reverse primers specific to the respective genes in a total volume of 25 µl. Denaturation
and annealing were performed at 94 °C and 58 °C for 30 sec, respectively, while
extension was performed at 72 °C for 60 sec. The primers used are summarized in Table
1. The PCR products were analyzed by electrophoresis in 2% agarose gel and stained
with ethidium bromide. The PCR products were subcloned into the pGEM-T Easy
10
vector (Promega, Madison, WI, USA). After the nucleotide sequence of each cDNA was
confirmed, the cDNA were used as standards for real-time PCR. To quantify Egr-1 gene
expression levels, real-time PCR was performed with a fluorescence thermal cycler
(Light Cycler System, Roche) using 0.5 mM of each primer (Table 1). The fluorescence
of SYBR Green (Qiagen, Hilden, Germany) at 530 nm was recorded at the end of the
extension phase and analyzed using the Light Cycler Software (Version 3). The level of
mouse glyceraldehyde-3-phosphate dehydrogenase (Gapdh)
mRNA was also
determined as an internal control.
2.11. Statistical analysis
The results are expressed as means SD. Statistical analyses were performed using
ANOVA and Bonferroni’s test, with p < 0.05 being considered statistically significant.
11
3. Results
Brown adipocytes and stromal vascular cells (SVC) were isolated from the
mouse interscapular BAT. RT-PCR analyses revealed that the former expressed Y1 and
Y5, but not Y2, types of NPY receptors, while the latter mainly expressed Y1 NPY
receptor (Fig. 1). To evaluate the effects of NPY on brown adipocytes, we measured in
vitro oxygen consumption rate of isolated adipocytes as an indicator of thermogenic
function. Oxygen consumption rate was 2.9 ± 0.7 nmol O2/min/105 cells before
stimulation and increased to 15.0 ± 4.3 nmol O2/min/105 cells after stimulation with NE
at 1 M and 10 M (Fig. 2A). When stimulated with NPY (0.01-1 M), oxygen
consumption rate did not change from the basal rate (Fig. 2B). When simultaneously
stimulated with NPY (0.1 M) and NE (1 M), oxygen consumption rate did not
change from the NE-enhanced rate (Fig. 2C). NPY also failed to modify the
NE-enhanced rate even at the sub-maximal concentration of NE used (0.1 M, Fig. 2D).
Next, we measured cAMP formation of isolated adipocytes in the presence of a
phosphodiesterase inhibitor. cAMP accumulation was 15.9 ± 11.0 pmol/105 cells before
stimulation and increased to 125.8 ± 1.9 pmol/105 cells after stimulation with NE at 1
M (Fig. 3A). When stimulated with NPY (0.1 M) in the absence or presence of NE
stimulation, cAMP accumulation did not change from the basal or NE-increased level,
respectively (Fig. 3A). Furthermore, when examining NPY-induced ERK signaling
pathway as reported previously [14, 16, 19, 34], NPY failed to activate activity-related
site-specific phosphorylation of ERK (Thr202/Tyr204) in isolated adipocytes while NE
stimulated it (Fig. 3B). Therefore, it is unlikely that NPY controls basal and
NE-regulated thermogenesis, although mRNAs of NPY receptors were expressed.
To evaluate the effects of NPY on SVC, we examined whether NPY induced
12
ERK phosphorylation. As shown in Figure 4, treatment of cells with NPY (30 nM and
100 n) induced the phosphorylation, indicating that functional NPY receptor was
present in SVC. We further examined STAT3 phosphorylation at Ser727 and Tyr705, as
the serine residue of STAT3 is known to be a downstream target of the ERK pathway
[15, 17, 22, 24, 39]. NPY treatment at the concentration of 30 nM or higher induced the
Ser phosphorylation, but not the Tyr phosphorylation (Fig. 5). In time course analyses,
NPY induced ERK phosphorylation in 3 min after the stimulation, which ended at 30
min, while NPY induced and ceased STAT3 phosphorylation at Ser727 at 10 min and
30 min after the stimulation, respectively (Figs. 4 and 5). Prior treatment of PD98059,
an ERK kinase (MEK) inhibitor, suppressed both phosphorylation of ERK and STAT3
at Ser727 (Fig. 6). Moreover, treatment of SVC with NPY increased the immediate
early gene, Egr-1 (early growth response 1, a STAT3-responsive gene) mRNA
expression at 1 h after the stimulation, and prior treatment of PD98059 suppressed the
expression (Fig. 7).
To evaluate the role of NPY in the regulation of SVC function, we examined
whether NPY induced cell growth since NPY promotes proliferation of adipocyte
precursor cells in visceral adipose tissue [42]. In contrast to the report, NPY failed to
stimulate cell growth of stromal vascular cells of BAT (Fig. 8).
13
Discussion
In the present study, we have demonstrated for the first time that NPY fails to
affect the basal and NE-enhanced cAMP production, ERK phosphoryllation and
oxygen consumption of brown adipocytes in vitro. These phenomena are inconsistent
with the previous findings that NPY has a suppressive role in the BAT thermogenesis
when given intraperitoneally [30]. As central activation and suppression of NPY
neuron decrease and promote BAT function, respectively [6, 8, 21], this inconsistency
may be explained by the assumption that intraperitoneally administered NPY leads to
activation of central NPY neurons. However, central action induced by peripheral NPY
remains to be elucidated. Our observation also conflicts with the reports showing that
NPY antagonizes -adrenergic agonist-induced activation of adenylyl cyclase [27, 36].
As mRNAs of Y1 and Y5 receptors coupled to Gi protein were detected in brown
adipocytes, suppression of cAMP production and prevention of increase in oxygen
consumption were simply expected. However, the failure of NPY to modulate the
cAMP production, oxygen consumption and also ERK activation suggests that
functional NPY receptors are lacking in brown adipocytes. Therefore, it is unlikely that
NPY directly controls basal and NE-regulated thermogenesis in a short period of time,
even if some sympathetic nerves carrying both NE and NPY innervate brown
adipocytes [23, 28, 38].
In contrast to brown adipocytes, we have demonstrated that NPY activates ERK
phosphorylation in stromal vascular cells (SVC) from the BAT. NPY has been shown to
activate ERK pathway in a variety of cells, possibly leading to cell proliferation of
neuronal precursor cells [14, 16] and progression of cancer [34]. Moreover, Yang et al.
show that NPY promotes ERK-dependent proliferation of adipocyte precursor cells
14
from the white adipose tissue [42]. We also observed NPY induction of Egr-1 gene, a
transcription factor involved in cell proliferation and in the regulation of apoptosis [12,
13]. It is thus assumed that NPY released from sympathetic nerve terminal stimulates
proliferation of SVC, resulting in hyperplasia of the BAT, which occurs in response to
physiological stimuli such as cold exposure [20]. However, NPY failed to promote
proliferation of SVC from the BAT. The difference in cellular response to NPY in
adipocyte precursor cells between the white and brown adipose tissues may be simply
due to the different characteristics of these cells which expressing different genes.
STAT3 is a transcription factor and activated upon phosphorylation of the tyrosine
residue at amino acid 705 by Janus-activated kinase (JAK) upon the stimulation of a
variety of cytokine receptors [4]. In cultured SVC, STAT3 at Tyr705 was constitutively
phosphorylated by unknown mechanisms. As transformation of fibroblasts by viral Src
induces constitutive activation of STAT3 at Tyr705 [4, 7], non-receptor tyrosine kinases
such as cellular Src (c-Src) might be involved in the constitutive activation of STAT3 in
the cells used. Under these conditions, we have demonstrated for the first time that NPY
induced phosphorylation of STAT3 at Ser727. It is likely that ERK is a STAT3 kinase,
since ERK activation preceded phosphorylation of STAT3 at Ser727 and the MEK
inhibitor abrogated the phosphorylation of both ERK and STAT3 in SVC, as reported
previously [15, 17, 22, 24, 39].
Tyrosine phosphorylation of STAT3 plays pivotal roles in its nuclear translocation
and transcriptional activating function [4, 31], but it is a fact that the Y705F mutant of
STAT3, which cannot be phosphorylated on tyrosine, also activates some gene
expression [41]. There are also conflicting reports regarding the significance of the
serine phosphorylation of STAT3 in its transcriptional activating function [10]. That is,
15
STAT3 phosphorylation at Ser727 has been shown to reduce and increase the
transcriptional potential of STAT3. In either case, our result that NPY enhanced a
STAT3-responsible gene (Egr-1) expression in a MEK-dependent manner suggests the
possibility that NPY modulates ERK- and STAT3-dependent transcription via its Ser727
phosphorylation. In addition, NPY is known to control various genes including
neuropeptide precursors [1, 32]. Therefore, STAT3 may be important in NPY-dependent
transcriptional regulation.
In summary, the present results suggest that NPY acts on SVC in BAT and plays
roles in the regulation of their gene transcription through ERK and STAT3 pathways,
while NPY does not affect the thermogenic function of brown adipocytes.
16
Acknowledgements
This work was supported by grants from the Japan Society for the Promotion of Science
(JSPS) to K.K. The work was also supported by JSPS Research Fellowships for Young
Scientists to K.S.
17
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22
Table 1. Primer list
Gene Forward primer sequence (Accession No.) Reverse primer sequence
(PCR product size)
Npy1r 5`- TCA ACA GAG GTG AAC AGA CG -3` (NM_010934) 5`- TCA ATG CCA GGT TTC CAG AG -3`
(+69 to + 429, 361 bps) Npy2r 5`- ATG GGC CAG GGC ACA CTA CT -3` (NM_008731) 5`- GGG TGT TCA CCA AAA GAT CC -3`
(+339 to + 581, 243 bps)
Npy5r 5`- CAT GCC ATT CCT TCA GTG TG -3` (NM_016708) 5`- TCT GGA ACG GTT AGG TGC TT -3`
(+550 to + 1140, 591 bps)
egr1 5'- TGG CCT GAA CCC CTT TTC AG -3' (NM_007913) 5'- GTA GAT GGG ACT GCT GTC GT -3'
(+721 to + 885, 165 bps)
Gapdh 5'- GAA GGT CGG TGT GAA CGG ATT -3' (NM_008084) 5'- AAG ACA CCA GTA GAC TCC ACG A -3'
(+56 to + 349, 294 bps)
23
Figure legends
Fig. 1 Expression of NPY receptors.
Conventional RT-PCR was performed on the mRNA obtained from mouse brain,
stromal vascular cells (SVC) and brown adipocytes (BA) isolated from mouse
interscapular BAT in order to detect expression of NPY receptors, Y1, Y2, and Y5.
Fig. 2 Norepinephrine (NE) increased oxygen consumption of isolated brown
adipocytes, but NPY failed to modify basal and NE-enhanced oxygen consumption.
Oxygen consumption by brown adipocytes was monitored in vitro for 20 min, and
increasing concentrations of NE (A) and NPY (B) and their combination (C, D) were
added at 5 min after the onset of the monitoring (arrows). Data are mean ± SD of values
from 3 independent experiments.
Fig. 3 Norepinephrine (NE) increased cAMP formation and ERK phosphorylation
in isolated brown adipocytes, but NPY failed to modify basal and NE-induced
cAMP formation and ERK phosphorylation. Brown adipocytes were stimulated with
NE (1 µM) and/or NPY (100 nM) for 10 min in the presence (A) and absence (B) of a
phosphodiesterase inhibitor. Then, in A, cAMP levels were determined. In B,
immunoblot analyses were performed for total and phosphorylated ERK. Data are mean
± SD of values from 4 independent experiments in A and from 8 independent
experiments in B, respectively. *p<0.05 vs. without NE treatments.
Fig. 4 NPY increased phosphorylation of ERK in stromal vascular cells of BAT in
dose- and time-dependent manners. Cultured stromal vascular cells were stimulated
24
with increasing concentrations of NPY (0.1-100 nM) for 10 min in A and 100 nM NPY
for 3-60 min in B. Immunoblot analyses for total and phosphorylated ERK were
performed on 20 µg of protein of each sample, and representative blots are shown. Data
are mean ± SD of values from 3 independent experiments. *p<0.05 vs. control (0 nM or
0 min).
Fig. 5 NPY increased phosphorylation of STAT3 at Ser-727, but not Tyr705, in
stromal vascular cells of BAT in dose- and time-dependent manners. Cultured
stromal vascular cells were stimulated with increasing concentrations of NPY (0.1-100
nM) for 10 min in A and 100 nM NPY for 3-60 min in B. Immunoblot analyses for total
and phosphorylated STAT3 were performed on 20 µg of protein of each sample, and
representative blots are shown. Data are mean ± SD of values from 3 independent
experiments. *p<0.05 vs. control (0 nM or 0 min).
Fig. 6 PD98059 abrogated both NPY-induced phosphorylation of ERK and STAT3
at Ser727 in stromal vascular cells of BAT. Stromal vascular cells in culture were
stimulated with NPY (100 nM) for 10 min, in the presence of either DMSO (-) or
PD98059 (5-50 µM), an ERK kinase inhibitor. Immunoblot analyses for total and
phosphorylated ERK (A) and total and phosphorylated STAT3 (B) were performed on
20 µg of protein of each sample. Representative blots are shown. Data are mean ± SD of
values from 4 independent experiments. *p<0.05 vs. without NPY treatment. †p<0.05
vs. without PD98059 treatment.
Fig. 7 NPY increased Egr1 expression in stromal vascular cells of BAT, which was
25
inhibited by PD98059. Stromal vascular cells in culture were stimulated with NPY
(100 nM) for 1 h, in the presence of either DMSO (-) or PD98059 (+, 50 µM). The
mRNA expression of Egr-1, known as a STAT3-responsive gene, was visualized by
conventional RT-PCR and quantified by real-time PCR. Data are mean ± SD of values
from 3 independent experiments. *p<0.05 vs. without NPY treatment. †p<0.05 vs.
without PDD98059 treatment.
Fig. 8 NPY failed to stimulate cell growth of stromal vascular cells of BAT. Stromal
vascular cells (5 × 103 cells) were cultured in 96-well plates with DMEM containing 1%
FCS and increasing concentrations of NPY for 72 h. Then, the number of cells was
assessed and expressed as a relative value to the control cells without NPY treatment (0
nM). Data are mean ± SD of values from 7 independent experiments.